The emergence of antibiotic resistance is a persistent problem that affects all available antibiotic treatments and poses a serious risk to human health. Current research in the van der Donk lab is focused on the discovery of robust antibiotics that derive from ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. In particular, much of this research centers on the lanthipeptide class of RiPPs, which are characterized by thioether-containing rings known as lanthionines. Studies on these peptides were spurred by the remarkable activity of nisin, a lanthipeptide that has been used as a food preservative for decades without the development of resistance. The basic enzyme-catalyzed post-translational modifications required for lanthipeptide biosynthesis are known; however, the mechanism of the enzyme activation necessary to trigger these transformations has not been elucidated. The majority of RiPPs arise from linear precursors containing leader peptides thought to play a critical role in enzyme activation for post-translational modifications, but the details of leader peptide-enzyme binding have not been determined for any RiPP. One goal of the proposed research is to characterize substrate-enzyme interactions for two classes of lanthipeptide natural products. First, the leader peptide binding site will be determined for class I modification enzymes nisin dehydratase (NisB) and nisin cyclase (NisC). There are two proposed models for leader peptide-enzyme binding involving either a dynamic or a static interaction. Results from the proposed studies will assign the mechanism of this interaction for nisin biosynthesis. Second, the first lanthipeptide precursor-enzyme co-crystal structure will be accessed by forming a stable bisubstrate analogue with the dehydratase domain of a class II modification enzyme. Both proposed studies take advantage of photochemical cross- linking, a powerful approach in both its precision and broad applicability. Photochemical cross-linking will be further employed in target identification for the antibiotic sublancin, a recently characterized S-linked glycopeptide RiPP featuring exceptional stability and an unusual structural motif. Despite extensive research on RiPPs, the proposed goals have only recently become accessible due to exciting advances in lanthipeptide research, photochemical cross-linking, and peptide preparation. Conclusions gained through mechanistic studies on lanthipeptide biosynthesis will be invaluable for further research on a wide variety of RiPPs with diverse biological properties. Most importantly, target identification and elucidation o the mechanistic details for lanthipeptide biosynthesis will both help to guide the continued pursuit of improved pharmaceuticals including more robust antibiotics.